Ion-exchange chromatography is a frequently used chromatography technique that separates ions and polar molecules based on their charge. It can be used for almost any type of charged molecule including large proteins, polypeptides, nucleic acids, polynucleotides, small nucleotides and amino acids. The surface of the stationary phase displays ionic functional groups, which interact with analyte ions of opposite charge through columbic (ionic) interactions. Ion-exchange chromatography is thus divided into cation-exchange chromatography and anion-exchange chromatography. In cation-exchange chromatography, positively charged cations are retained because the stationary phase displays a negatively charged functional group, whereas in anion-exchange chromatography, anions are retained by positively charged functional groups on the stationary phase.
Proteins are ampholytes in that they generally have both negative and positive charges. For example, aspartic acid and glutamic acid residues display negative charges while arginine, lysine and histidine residues exhibit positive charges. The net charge on a protein, however, is often dependent on the composition of charged residues as well as the pH of the environment the protein is in.
Nonporous polymeric and silica particles typically have low surface areas, which is the main factor requiring the use of surface polymerization techniques to produce ion-exchange stationary phases based on nonporous particles. For instance, surface polymerization can be used to modify column capacities through incorporation of multiple ion-exchange groups into long polymeric chains, which are attached to the surface of the nonporous particles.
Nonporous substrates have been used for separation of biomolecules and monoclonal antibodies, in particular, due to unrestricted access of biomolecules to ion-exchange sites located at the surface of nonporous particles. However, surface functionalization via surface grafting, such as by polymerization, can be affected by many factors, including temperature, concentration of oxygen, mass-transfer during polymerization process, which can contribute negative effects on reproducibility of products. Also, commercial nonporous materials are not available in all desired particle sizes, which limits their use to certain available column formats.
A further disadvantage is that high back-pressure can develop in ion-exchange columns packed with nonporous particles, which are typically coupled with long polymeric chains attached to the particle surface. High back-pressure can become pronounced in columns packed with such nonporous particles, especially if they are small in size.
There remains an unmet need for stationary phase materials and compositions and related ion-exchange chromatographic methods that provide improved separation characteristics and efficiency as well as allow broader applications.